Focus forming unit assay LLC-MK2

Focus forming unit assay LLC-MK2 download the handbook target cells were seeded at a density of 1��105 cells in each well of a 6-well plate 24 h prior to infection. Approximately 200 focus forming units (FFU) of virus were incubated with or without peptide in serum-free DMEM for 1 h at rt. Virus/peptide or virus/control mixtures were allowed to infect confluent target cell monolayers for 1 h at 37��C, with rocking every 15 m, after which time the medium was aspirated and overlaid with fresh DMEM/10% (v/v) FBS containing 0.85% (w/v) Sea-Plaque Agarose (Cambrex Bio Science, Rockland, ME) without rinsing. Cells with agar overlays were incubated at 4��C for 20 m to set the agar. Infected cells were then incubated at 37��C with 5% CO2 for 5 days.

Infected cultures were fixed with 10% formalin overnight at 4��C, permeablized with 70% (v/v) ethanol for 20 m, and rinsed with phosphate buffered saline, pH 7.4 (PBS) prior to immunostaining. Virus foci were detected using a specific mouse mAb from hybridoma E60 (obtained from M. Diamond at Washington University), followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Pierce, Rockford, IL), and developed using AEC chromogen substrate (Dako, Carpinteria, CA). Results are expressed as the average of at least two independent trials with three replicates each. IC50 values were determined using variable slope sigmoidal dose-response curve fits with GraphPad Prism 4.0 software (LaJolla, CA), except for DN81opt, which was determined graphically due to a lack of data points to produce a reasonable curve fit.

Toxicity assay Cytotoxicity of peptides was measured by monitoring mitochondrial reductase activity using the TACS? MTT cell proliferation assay (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. Dilutions of peptides in serum-free DMEM were added to confluent monolayers of LLC-MK2 cells in 96-well plates for 1 h at 37��C, similar to the focus forming inhibition assays, and incubated at 37��C with 5% (v/v) CO2 for 24 h. Absorbance at 560 nm was measured using a Tecan GeniosPro plate reader (Tecan US, Durham, NC). Cryoelectron microscopy DENV-2 NGC strain used for the cryoEM reconstructions was propagated in mosquito C6/36 cells. Virus was purified by precipitation with 40% PEG 8000 and then ultracentrifugation onto a 25% sucrose cushion.

Virus was further purified by banding on a 10%�C30% potassium tartrate gradient. The virus band was removed and dialyzed against 12 mM Tris pH 8.0, 120 mM NaCl, 1 mM EDTA, and concentrated using a Millipore Centricon filter. Purified virus was mixed with 1OAN or DN57opt at a concentration Entinostat of 1 molecule of peptide for every E protein on the surface of the virus. The complex was incubated for half an hour at 37��C followed by half an hour at 4��C and then flash frozen on holey carbon grids in liquid ethane.

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